Mycobacterial disease, primarily tuberculosis, kills nearly two million people annually. Ineffective vaccines, as well as multi-drug and extremely-drug resistant strains of M. tuberculosis, exacerbate this chronic global crisis. Clearly, new efficacious drugs are needed to fill the growing therapeutic void. Rational drug design begins with a foundation of genomic sequence information. Building upon this genomic foundation requires meaningful annotation of gene product activity and, more importantly, how the encoded proteins and the pathways they comprise come together to produce a viable mycobacterium. Screens have been developed in model organisms to connect genes by epistatic interactions on a genomic scale. Two mutations may be viable separately, but lethal when brought together. Such synthetic lethal interactions link the respective gene products together in an essential process. The power of synthetic genetic analysis grows exponentially with the number of genes analyzed, so testing many non-essential genes in a synthetic genetic array (SGA) is ideal. Hundreds of thousands of synthetic interactions have been described in yeasts and E. coli, and typically a single gene displays ~30 interacting partners. Large-scale approaches require a genetically tractable organism to easily create individual mutants, a process to efficiently combine individual mutations, and a reliable assay, all amenable to a high-throughput format. Mycobacterium smegmatis meets all of these requirements and will form the basis for the first comprehensive mycobacterial SGA (mSGA). Even though all of the essential components are in place, evaluating the feasibility of the approach and the establishment of a functional pipeline are prerequisites to initiating a full mSGA. We will use conjugal DNA transfer to combine individual mutations in M. smegmatis. An inability of these double mutants to grow will indicate synthetic lethality, linking the two mutant genes. We will perform limited screens progressing from candidate gene pairs with known synthetic lethality, to a more complex system with both known and likely unknown interactions, and finally with representative hypothetical genes with no known activity or interaction. This progression will provide the necessary positive controls to show that the system is working as intended, and it provides an opportunity to assess the potential wealth of information that an expanded mSGA could yield. Integration of epistatic information from an expanded mSGA with other meta-data from proteomic and transcriptional studies will provide a systems biology view of a mycobacterium. This application will establish an mSGA pipeline, and will identify a preliminary set of synthetic lethal interactions as proof-of-principle for its potential. Synthetic lethal interactions, identified using an unbiased genome-wid approach, identify essential functions that represent new high-value potential drug targets.